This invention pertains generally to the field of microscopic spectroscopy such as Raman spectroscopy.
When illuminating light, such as a laser beam, is incident upon a sample material, molecular bonds in the material will be excited by the incident light and will emit radiation which can be detected as scattered light. The Raleigh component of the scattered light corresponds to the light emitted when the molecule relaxes from the excited state to the ground state. Infrequently, the molecule will relax to a different vibrational or rotational level in the ground state. This produces Raman scattering components at Stokes and Anti-Stokes frequencies. A sample composed of multiple molecular species will produce a spectrum of such Raman scattering. The Raman scattering components can be detected and analyzed to help determine the composition of the sample. Various apparatus have been developed for analyzing Raman spectra including Raman microscopes in which a very small area on a sample can be analyzed to determine characteristics of the composition of the sample at that area. In a typical Raman microscope, narrow band or monochromatic illuminating light, such as from a laser, is passed along a beam path through the objective lens of the microscope where it is focussed at a focal point on a specimen. The Raman scattering from the sample collected by the microscope objective is passed back on a beam path to a spectrograph which typically separates the Raman scattering radiation by wavelength and detects it. Optical elements are typically included in the excitation beam path and the returning Raman radiation beam path to separate the excitation light from the Raman scattering light and to filter out the Raleigh light from the beam directed to the spectrograph. The microscope may also include a wide field illumination beam path in which light from the microscope is passed up on a beam path to a position where it can be viewed directly by an operator or detected by a video camera for display to an operator.
In using a Raman microscope of this type, the operator will generally view the specimen through the microscope to select a small area in the specimen to be analyzed, generally by aligning the desired area to be analyzed in the center of the microscope visual field, typically with the aid of cross hairs or other indicia. The intention is that the illuminating light beam path will have its axis aligned with the visual beam path so that the illuminating light will be focussed onto the spot in the specimen that was targeted by the operator using the microscope. However, if the illuminating light beam is not, in fact, aligned with the axis of the visual microscope beam path, the illuminating light will impinge on the sample at a different position, leading to potentially erroneous data concerning the composition of the sample. In addition, if the returning beam path which includes the Raman scattering radiation is not properly aligned with the aperture of the spectrograph, the intensity of the (already very weak) Raman scattering radiation received by the spectrograph will be reduced. This loss of signal power can give rise to corruption of the spectrographic data by noise and a reduction of the full potential of the spectroscopic microscope to obtain information concerning the composition of the sample.
All Raman microscope systems are subject to minor alignment drift over time. These drifts can be caused, for example, by environmental changes due to temperature fluctuations and external vibration, and by normal wear of components occurring during operation. Over time, the performance of the Raman microscope system degrades and the misalignments must be corrected by periodic maintenance procedures. Such maintenance is frequently difficult because of the complexity of simultaneously aligning the various beam paths, and generally requires trained maintenance personnel. This maintenance is thus costly both because of the direct expense associated with the maintenance procedures and because of the down time of the microscope during the maintenance procedures.
In accordance with the present invention, alignment of the multiple beam paths in a spectrographic microscope such as a Raman microscope is carried out rapidly and efficiently, without the need for trained maintenance personnel, and with minimal operator involvement. The alignment procedure may be carried out under software control by the microscope system computer. Because the alignment process is quickly and easily carried out, realignment can be performed much more frequently with minimal instrument down time, allowing the instrument to be maintained at peak performance levels.
In carrying out the invention, the operator of the microscope uses the visual microscope to position an aperture at an intended focal point of the microscope. This aperture can comprise an entrance aperture for a detector, an exit aperture for a source, or both. After the operator has fixed the aperture at the intended focal point, the spectroscopic microscope system may then be operated to either detect light using the spectrographic detector that exits from the aperture or to project illuminating light through the microscope to be focussed at the aperture and detected. The illuminating light beam path may then be adjusted to maximize the intensity of the light passed through the aperture and detected, and the return beam path leading to the spectrograph may be adjusted to maximize the light exiting from the aperture that is detected by the spectrograph. The maximization of the detected light in the illuminating beam path ensures that the illuminating beam is maximally aligned to focus on the intended focal point, whereas maximization of the detected light in the return beam path will result in the maximum return of Raman scattered light from a specimen that emanates from the focal point of a sample mounted on the microscope.
An alignment instrument that may be utilized in accordance with the invention comprises a housing having a plate with a spatially limited aperture therein, e.g., a pinhole aperture. A light source is mounted within the housing and is selectively activatable to project light out of the housing through the aperture. A light detector is also mounted in the housing to detect light originating outside the housing that passes through the aperture. The detector provides an output signal which corresponds to the intensity of the detected light. The alignment instrument is preferably formed so that it is readily mountable on the stage of the microscope, which can be adjusted until the operator, using the visual system of the microscope, observes the aperture of the alignment instrument located at the desired focal point of the microscope objective lens. Indicia may be provided in the microscope in a conventional manner to help locate the focal point. The alignment instrument may then be used without moving it from that position to project light out from the aperture to be detected by the spectrograph detector for maximization of the return beam path and, separately, to detect light from the illuminating beam that is focussed onto the focal point and passed through the aperture to allow adjustment of the illuminating beam to properly align it.
In the present invention, means are preferably provided in the illumination beam path to adjust the axis of the beam to allow it to be aligned to the aperture of the alignment instrument. Similarly, adjustment means are preferably provided in the return beam path to allow adjustment of the axis of the return beam to best fit the return beam to the input aperture of the spectrograph. A preferred structure for such adjustment means includes a pair of sequential lenses in the beam path which are adjustable relative to one another in two dimensions. Preferably, the pair of lenses have focal lengths of equal magnitude but opposite sign. One lens may be fixed and simply compensates for the other to produce a system with negligible optical power. The adjustable lens can be displaced from the optical axis in two dimensions, so that the pair of lenses can be used to effectively introduce an angular deviation to the beam passing through it. The motion of the adjustable lens may be controlled by motor drives in the two dimensions.
The alignment process in accordance with the invention may be carried out automatically under computer control. The microscope system may be programmed to periodically prompt the operator to perform alignment or it may provide a prompt when the need for realignment is detected. The operator places the alignment instrument on the stage of the microscope and adjusts the centration and focussing of the microscope using the oculars or a video display to position the aperture of the alignment instrument at the desired focal point of the microscope. This alignment procedure defines the microscope axis. After this procedure has been performed by the operator, the operator enables the system to carry on the alignment process automatically. The system turns on the alignment instrument light source and adjusts the spectrograph beam path, utilizing the adjustment means, to maximize the intensity of radiation entering the spectrograph. The system then turns off the light source in the instrument, and turns on the illuminating light source in the microscope, typically a laser. The system then adjusts the illumination beam path using the illumination beam adjustment means to maximize the intensity of radiation passing through the aperture of the alignment instrument and that is detected by the detector within the instrument. After these processes are completed, the system then can provide a message to the operator that alignment has been completed and that the alignment instrument may be removed from the microscope stage.
Further objects, features and advantages of the invention will be apparent from the following detailed description when taken in conjunction with the accompanying drawings.